Project Summary/Abstract The internal state of arousal can dramatically influence behavior, from sensory processing to cognitive and emotional function. Disrupted arousal is a symptom common to several psychiatric disorders, including depression, anxiety, addiction, attention deficit hyperactivity disorder, and schizophrenia. Arousal can change over multiple timescales, including rapid fluctuations that optimize performance during cognitive functions. Slower forms of arousal are linked to the activity of multiple neuromodulatory cell types, including those releasing monoamines, acetylcholine, and numerous peptides; conversely, rapid arousal has been primarily attributed to the noradrenergic locus coeruleus. Neuromodulators are challenging to investigate in behaving mammals, because they are small, deep, spatially dispersed, and molecularly diverse; consequently, a comprehensive survey of neuromodulatory systems underlying rapid arousal has not been conducted. I propose to overcome these obstacles by developing and applying tools to study neuromodulation and arousal in larval zebrafish. These vertebrates share conserved neuromodulatory systems with mammals, yet are small and transparent, so the neuromodulatory cell types underlying fast-timescale arousal can be exhaustively mapped at the scale of the whole brain using cellular-resolution functional imaging. I hypothesize that multiple neuromodulatory systems act in parallel to implement fast-timescale arousal. The goal of this proposal is to identify and characterize the neuromodulatory systems implementing the internal state of arousal, and determine how these systems shape global neural dynamics. In preliminary efforts, I developed a novel whole-brain cellular-resolution tissue registration method for aligning the same neurons from live activity recordings with postmortem immunohistochemical identification of multiple neuromodulatory cell types. In the K99 mentored phase, I will use this method to catalogue the neuromodulatory cell types correlated with trial-to- trial fluctuations in arousal, measured by sensorimotor reaction times. My preliminary data have revealed multiple noradrenergic, cholinergic, serotonergic, dopaminergic, and peptidergic populations correlated with arousal. I will subsequently map the functional connectivity of these arousal-correlated populations by combining brain-wide imaging with optogenetics in transgenic fish, through training with my mentor Dr. Karl Deisseroth and co-mentor Dr. Philippe Mourrain. In the R00 independent phase, I will apply these skills to determine the causal impact of arousal-correlated neuromodulatory cell types on brain-wide dynamics and the behavioral expression of arousal. Comprehensive training with Dr. Deisseroth and Dr. Mourrain at Stanford University will provide me with the skills required to pursue research related to arousal and other internal states as an independent investigator. These efforts will lead to insights into a class of arousal dysfunction symptoms common to a diverse array of psychiatric disorders.